The present invention relates to protease inhibitors, specifically to cystatins, that have been modified by glycosylation in order to enhance stability and activity; to methods of making such modified protease inhibitors and to methods of using such modified protease inhibitors to inhibit proteolysis of a protein substrate.
Proteases are enzymes that degrade proteins. Proteases are classified by the substrate upon which they act and include serine proteases, cysteine proteases, aspartate proteases and metalloproteinases. Serine and cysteine proteases are widespread and are found in diverse organisms including eukaryotic and prokaryotic animals and plants. Cysteine proteases are generally well characterized enzymes having a known primary structure composed of alpha helices and beta pleated sheets (8).
Proteases mediate many processes that are harmful to man, either by producing pathology or by causing economic loss, for instance by degrading foods. Protease-mediated pathology is known to be caused by a wide variety of organisms including bacteria, such as staphylococci and streptococci, fungi, arthropods, nematodes, protozoa such as amoebas, intestinal flagellates, haemoflagelates, such as Leishmania and trypanosomes and helminths.
Proteases are known to be important in the pathology of certain viruses (9, 11, 12, 31) including Polio virus, Herpes virus, Corona virus, HIV and Rotavirus. Proteases are also known to play a role in various diseases with no clear etiological agent, such as muscular dystrophy (7) and cancers (1) including breast cancer (2), and amyloid angiopathy, a genetic disease that often leads to fatal cerebral hemorrhages in young adults (10, 13).
Proteases are also responsible for the spoilage of economically important foodstuffs, necessitating huge annual expenditures on preventative measures. For instance, the fungus Botrytis cinera causes widespread disease in over thirty species of commercial crop plants. Molds and fungi are the major destroyers of citrus fruit crops. Foods rich in protein, such as meat and fish, are degraded and made inedible by proteases from Pseudomonads and other bacteria. Foods with a high muscle content may be quickly broken down by endogenous proteases released from tissues upon death. These enzymes degrade myosin, and destroy the texture of the food. An important example of such spoilage occurs during the processing of surimi, which is a form of processed minced fish, commonly made from Pacific Whiting (Merluccius productus), and is the main ingredient of seafood analogs such as xe2x80x9cimitation crab meatxe2x80x9d. Surimi is an important source of relatively cheap, high quality, low fat protein important to the diet of many people in the Far East and of increasing economic significance worldwide. Endogenous proteolytic enzymes released during surimi production cause rapid degradation of muscle tissue and lead to poor quality surimi (3, 4, 5, 6, 11, 17, 18, 19). It is thought that the protease released from the fish tissue is a cathepsin, which is a common cysteine protease (3).
Because of the role of proteases in these various processes, protease inhibitors have been investigated for their potential role in preventing disease and degradation of foodstuffs (4, 5, 18). Partially refined substances that contain protease inhibitors are commonly used in food processing, for instance, to prevent proteolytic breakdown of fish protein during the production of surimi (4, 17, 18). The most commonly used food-grade protease inhibitors are beef plasma protein (BPP), egg white powder and potato powder (4). Genetic engineering techniques have been used to introduce protease-inhibitor genes from chickens into cereal and grass plants to control protease-producing plant pathogens. Likewise, a plant protease inhibitor gene, from Cowpea, has been recombinantly introduced into tobacco, tomato, cotton and other plants to inhibit destruction by nematode worms (16).
Cystatins are cysteine protease inhibitors that are members of Family 2 of the cystatin superfamily, characterized by a single chain of about 115 to 122 amino acids with a molecular weight of about 13000, having two disulfide bonds (7, 8, 10). Cystatins and protease enzymes such as cathepsins form tight (but reversible) enzyme-inhibitor complexes with dissociation constants typically in the nannomolar range (10).
A number of cystatins have been characterized including human cystatins (C, S, SN, SA, D, M and E), mouse cystatin, egg-white cystatin, bovine cystatin, carp cystatin, trout cystatin and salmon cystatin. In their natural state, cystatins protect the body by inhibiting the potentially harmful effects of proteolysis, and may prevent destruction of connective tissue by protease enzymes, for instance, lysosomal proteases, released from dying or damaged cells (16).
Cystatins have been investigated for potential medical applications, for instance, to inhibit replication and pathology of Picornaviruses (12), Coronaviruses (9) and Herpes Simplex type 1 virus (15, 16, 30). Cystatins may also play a natural role in prevention of bacterial infection by E. coli, Shigella (13), Leishmania, Schistosoma and Entamobea (10) which appear to use proteases to facilitate tissue invasion.
Cystatins are likely the primary protease inhibitors in food-grade protease inhibitor preparations such as beef plasma protein and egg white powder. Since cystatins are themselves proteins, they are prone to denaturation and loss of activity when exposed to unfavorable temperatures or pH. Many food production processes, including surimi production, involve elevated temperatures (17). Presently, in order to maintain cystatin activity, more cystatin must be added after cooling. Adding additional cystatin is both labor-intensive and expensive. Also, when cystatins are used for medical treatment, either as a topical or ingested medication, it is preferable for the cystatin-containing composition to be sterile. A common method of sterilization involves treatment with elevated temperatures. A cystatin that could maintain activity despite exposure to elevated temperatures would thus be useful in food processing and in drug formulation.
The present invention provides modified, glycosylated, heat-stable cystatins and methods of making and using these cystatins. The present invention also provides nucleic acid molecules encoding such cystatins.
The nucleic acid molecules of the invention have been modified so that when such a nucleic acid molecule is expressed in a eukaryotic cell, certain amino-acid residues of the expressed cystatin protein are glycosylated during post-translational modification of the protein. The resulting mature protein has attached, at specific amino acid residues, sugar molecule chains of varying length. The present invention includes the nucleic acid molecules that encode modified cystatins based on the cystatins from humans (C, S, SN, SA, D, M and E), egg white, cow, carp, trout and salmon.
Various residues in the cystatin primary amino acid sequence have been identified where the introduction glycosylated residues increases heat stability of the expressed protein without severely affecting enzymatic activity. In human cystatin C, for instance, the sites for glycosylation include amino acid residues at positions 35, 36 and 79.
The present invention also includes a method of making modified heat-stable cystatins by modifying the nucleic acid molecules that encode cystatins. Such nucleic acid molecules are modified at certain defined sites and expressed in a eukaryotic cells. The present invention also includes a cell that contains at least one nucleic acid molecule encoding at least one modified, glycosylated, heat-stable cystatin. The cells of the invention may be of many types, for instance they may be cells from a yeast, a mammal, an insect, or a plant.
The invention also includes methods of inhibiting proteolysis of a protein substrate by contacting the protein substrate with a modified heat-stable cystatin having at least one engineered glycosylation site. Such a method may be applied, for example, to food processing, such as the production of surimi.
The invention also includes a method of treating a protease-mediated pathology of an organism, such as a mammal, a fish or a plant by administering to the organism a modified heat-stable cystatin of the invention. By such administration, the modified heat-stable cystatin contacts the protease that mediates the pathology, thereby inhibiting proteolysis by the protease and thereby treating the pathology.
SEQ ID NO: 1 shows the cDNA sequence and the amino acid sequence of native human cystatin C.
SEQ ID NO: 2 shows the amino acid sequence of native human cystatin C.
SEQ ID NO: 3 shows the cDNA sequence and the amino acid sequence of native human cystatin S.
SEQ ID NO: 4 shows the amino acid sequence of native human cystatin S.
SEQ ID NO: 5 shows the cDNA sequence and the amino acid sequence of native human cystatin SN.
SEQ ID NO: 6 shows the amino acid sequence of native human cystatin SN.
SEQ ID NO: 7 shows the cDNA sequence and the amino acid sequence of native human cystatin SA.
SEQ ID NO: 8 shows the amino acid sequence of native human cystatin SA.
SEQ ID NO: 9 shows the cDNA sequence and the amino acid sequence of native human cystatin D.
SEQ ID NO: 10 shows the amino acid sequence of native human cystatin D.
SEQ ID NO: 11 shows the cDNA sequence and the amino acid sequence of native human cystatin M.
SEQ ID NO: 12 shows the amino acid sequence of native human cystatin M.
SEQ ID NO: 13 shows the cDNA sequence and the amino acid sequence of native human cystatin E.
SEQ ID NO: 14 shows the amino acid sequence of native human cystatin E.
SEQ ID NO: 15 shows the cDNA sequence and the amino acid sequence of native egg white cystatin.
SEQ ID NO: 16 shows the amino acid sequence of native egg white cystatin.
SEQ ID NO: 17 shows the cDNA sequence and the amino acid sequence of native carp cystatin.
SEQ ID NO: 18 shows the amino acid sequence of native carp cystatin.
SEQ ID NO: 19 shows the cDNA sequence and the amino acid sequence of native salmon cystatin.
SEQ ID NO: 20 shows the amino acid sequence of native salmon cystatin.
SEQ ID NO: 21 shows the cDNA sequence and the amino acid sequence of native trout cystatin.
SEQ ID NO: 22 shows the amino acid sequence of native trout cystatin.
SEQ ID NO: 23 shows the cDNA sequence and the amino acid sequence of native bovine cystatin.
SEQ ID NO: 24 shows the amino acid sequence of native bovine cystatin.
SEQ ID NO: 25 shows the first of four oligonucleotides used to create a nucleotide coding for modified human cystatin C.
SEQ ID NO: 26 shows the second of four oligonucleotides used to create a nucleotide coding for synthetic human cystatin C.
SEQ ID NO: 27 shows the third of four oligonucleotides used to create a nucleotide coding for synthetic human cystatin C.
SEQ ID NO: 28 shows the fourth of four oligonucleotides used to create a nucleotide coding for synthetic human cystatin C.
SEQ ID NO: 29 shows a forward primer used in site-directed mutagenesis to introduce a glycosylation site at residue 35 of a modified human cystatin C.
SEQ ID NO: 30 is a reverse primer used in site-directed mutagenesis to introduce a glycosylation site at residue 35 of a modified human cystatin C.
SEQ ID NO: 31 is the forward primer used in site-directed mutagenesis to introduce a glycosylation site at residue 36 of a modified human cystatin C.
SEQ ID NO: 32 is the reverse primer used in site-directed mutagenesis to introduce a glycosylation site at residue 36 of a modified human cystatin C.